1. Field of the Invention
The present invention is in the field of recombinant genetics.
2. Related Art
The mechanism of cell death has come under increasing scrutiny in the last few years as scientists have sought to understand the biological basis of this phenomenon. Recent studies have shown that, in many cases, cell death is an active process involving a complex network of death triggers, death regulators and death effectors. The interactions of these death factors produce cell death pathways whose characteristics are really no different in principle from the pathways of intermediary metabolism. This realization has opened up the "black box" of cell death, allowing physiologists, biochemists and geneticists to begin to delineate cell death pathways and to identify the molecules involved.
Classically, two distinct forms of cell death have been recognized: necrosis and apoptosis. These processes have been distinguished based mostly on morphological criteria. In necrosis, cell and organelle swelling is a notable feature and the cells eventually burst releasing their contents into the surrounding tissue space. By contrast, the hallmark of apoptosis is cell shrinkage and organelle changes are uncommon. The exception to this is the nucleus where the chromatin condenses and becomes redistributed just under the nuclear envelope. Rather than bursting, apoptotic cells break up into small parcels containing plasma membrane, cytosol, organelles and pieces of nucleus. These so-called "apoptotic bodies" are eventually phagocytosed by neighboring cells, and can be visualized histochemically. Another important attribute of apoptosis (and the only established biochemical marker) is the discrete degradation of DNA resulting in a characteristic "DNA ladder" when analyzed on DNA-separating gels. These features (cell swelling vs. cell shrinkage and DNA ladder formation) have been used to characterize the death process in a variety of in vitro and in vivo models of cell death. However, it is important to realize that, although necrosis and apoptosis are still discussed in the literature as being distinct entities, they may, in fact, represent the extremes of a continuum of cell death processes and might share certain molecular features.
There are profound clinical implications to understanding the biology of cell death. In the normal development of a multicellular organism, many more cells are produced than are needed in the mature or adult tissue. Therefore, the process of cell birth (mitosis) must be counterbalanced by a process for reducing cell number. Obviously, if this homeostatic mechanism is disturbed, cells will die when they shouldn't and will live when they should die. There is a tremendous range of diseases in which a disruption of the normal cell death pathway may therefore play a central role. In nervous system disorders such as stroke and head trauma, neurons die due to external death triggers such as a lack of oxygen or excess excitatory neurotransmitters. Recent studies suggest that at least a portion of this neuronal death may occur by an apoptotic mechanism. In other neural diseases, such as Alzheimer's disease, and amyotrophic lateral sclerosis, neurons die as part of a degenerative process whose triggers have not yet been determined, but which could possibly involve apoptosis. By contrast, in disorders such as cancer and hyperimmune diseases, cells live when they should die, resulting in tumors (cancer) or an overactive immune system (autoimmune diseases). Thus, the ability to intervene pharmacologically in the cell death pathway may provide a completely new set of therapeutic agents useful in a wide range of human diseases.
Several gene families have been implicated as critical players in the cell death pathway One of the most interesting of these gene families is the bcl-2/ced-9 family. The first member of this family, bcl-2was initially identified as a causal factor in certain types of lymphatic cancer (B-cell lymphoma, hence the name). In this disorder, bcl-2 is overexpressed resulting in an abnormally longer lifespan for B-cells which allows these cells to accumulate additional mutations resulting in frank malignancy and lymphatic tumor development. Since being identified, the bcl-2 gene and its protein product have been intensively studied and found to be an effective blocker of both necrotic and apoptotic cell death in several model systems, including models of nerve cell death. The biochemical function of bcl-2 is not known (i.e., it is not clear whether it acts as an enzyme, receptor or signaling molecule). However, some experiments suggest that bcl-2 may play a role in cellular antioxidant pathways. This antioxidant theory of bcl-2 action is bolstered by the finding that the bcl-2 protein is localized to intracellular membranes, including mitochondria, an organelle thought to participate in the production of toxic oxygen radicals. However, it should be kept in mind that some experiments suggest that bcl-2 can block cell death even under conditions when oxygen radical-inducing death does not occur, suggesting that there may be several parallel pathways of cell death, with bcl-2 having the ability to block several of these pathways.
Since the discovery of bcl-2, nine other genes structurally related to it have been discovered (see Table 1). These genes include ced-9, a bcl-2 homologue found in the worm, C. elegans, and several related genes expressed in viruses. The existence of this gene family suggests that a group of bcl-2-like molecules function within the cell to control cell viability. Interestingly, not all bcl-2 family members block cell death. Instead, some seem to promote cell death. In addition, some members of the family can bind to other members of the family, and this interaction appears to regulate the cell death pathway. These findings have led to the hypothesis that the bcl-2 family members form part of a combinatorial mechanism. In this model, the interaction of family members with each other, and possibly with unrelated proteins, can determine whether a cell will live or die.
For a recent review of the bcl-2 family of proteins, reference is made to Davies, A. H., Trends Neuroscience 18:355-358 (1995). See also WO95113292, WO9500160 and U.S. Pat. No. 5,015,568.
Because of the importance of the bcl-2 family in controlling cell death, a PCR-based cloning strategy was employed to identify new members of this family with particular emphasis on finding bcl-2 homologues which are enriched in the brain. The present invention is directed to the molecular cloning and analysis of one such homologue, which is designated bcl-y.